The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins hen egg white lysozyme and a-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions call the a- and b domains. In EL and a-LA the latter domain specifically binds Ca+2. It is shown that, in contrast to HEWL in which the two domains unfold as a single cooperative unit, in EL the two domains unfold in two separate cooperative stages even in the presence of excess Ca+2. The thermodynamic characteristics of Ca+2 binding have been determined, and indicate that it is an entropically driven process. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca+2, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in EL is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the a and b domains.